Abstract

By combining targeted mutagenesis, computational refinement, and directed evolution, a modestly active, computationally designed Diels-Alderase was converted into the most proficient biocatalyst for [4+2] cycloadditions known. The high stereoselectivity and minimal product inhibition of the evolved enzyme enabled preparative scale synthesis of a single product diastereomer. X-ray crystallography of the enzyme-product complex shows that the molecular changes introduced over the course of optimization, including addition of a lid structure, gradually reshaped the pocket for more effective substrate preorganization and transition state stabilization. The good overall agreement between the experimental structure and the original design model with respect to the orientations of both the bound product and the catalytic side chains contrasts with other computationally designed enzymes. Because design accuracy appears to correlate with scaffold rigidity, improved control over backbone conformation will likely be the key to future efforts to design more efficient enzymes for diverse chemical reactions.

Diels–Alder reaction between 4-carboxybenzyl-trans-1,3-butadiene-1-carbamate (1) and N,N-dimethylacrylamide (2). The theozyme for promoting formation of the 3R,4S endo cyclohexene product (3) is shown in brackets. The phosphorylated product analog (4) was used for inhibition and crystallization experiments.

Characterization of Diels-Alderase CE20. (A) Dependence of reaction velocity on dienophile concentration at different fixed diene concentrations. (B) pH dependence of the enzymatic reaction. (C) Stereoselectivity of the uncatalyzed (background) and CE20-catalyzed reactions; authentic standards (control) are shown for reference. All error bars reflect the SD of three independent measurements.

Crystal structure of CE20 with bound product. (A) The structures of CE20 (orange), CE6 (gray), and the DA_20_00 computational design model (black) are very similar. The catalytic amino acids Tyr134 and Gln208 (shown as sticks) adopt nearly identical conformations. (B) The largest differences between CE20 and CE6 are localized in the appended lid element. The helix designed to interact with the substrates (left in the figure) overlays well with its counterpart in CE6, whereas the interhelical loop and the supporting helix (right) are largely disordered in CE20. (C) The reaction product (black carbons) binds deeply in a shape-complementary pocket (transparent gray surface) containing a buried water molecule (red sphere). (D) The catalytic residues interact with the ligand as designed. The side-chain phenol of Tyr134 donates a hydrogen bond to the carboxamide carbonyl group, whereas the side-chain amide of Gln208 accepts a hydrogen bond from the carbamate NH of the product and donates a hydrogen bond to the carboxamide carbonyl group. The latter interactions are offset by an unfavorable interaction between the Gln208 amide and the carbamate ether oxygen (red dashes). (E) Superposition of the product and catalytic residues shows that the crystal structure (black and yellow carbons) closely matches the original design model (gray and brown carbons). Hydrogen bonds in the design are depicted as black dashes. An unfavorable interaction between Gln208 amide and the carbamate ether oxygen is also evident in the design (3.2 Å in the design vs. 3.0 Å in the crystal structure).

Evolution of a shape-complementary pocket. Cut-away views of the active sites of DA_20_00 variants illustrate the gradual contraction of the binding pocket around the reaction product over the course of evolution. (A) DA_20_00, the original computational design, (B) the evolutionary intermediate CE6, and (C) CE20, the most evolved variant. The ligand depicted in the DA_20_00 and CE6 pockets (green carbons) was docked manually in the same orientation as the product seen in the crystal structure of the CE20 complex (black carbons). (D) Plot of effective molarity (EM = kcat/kuncat) vs. catalytic proficiency (1/KTS = [kcat/(Kdiene·Kdienophile)]/kuncat) for kinetically characterized catalytic antibodies (dark blue), a ribozyme (cyan), and descendants of the computationally designed DA_20_00 Diels-Alderase (red).